Enterococci are opportunistic pathogens that cause life-threatening infections in humans. An overarching goal of my research is to characterize mechanisms contributing to the evolution of antibiotic resistance in the enterococci. The proposed research will focus on Enterococcus faecalis, which since the 1980s has gained increasing notoriety from association with hospital-acquired infections and multidrug resistance including resistance to the last-line antibiotic vancomycin. Certain phylogenetic lineages of E. faecalis are associated with antibiotic resistance acquired through horizontal gene transfer and hospital endemicity - these are the high-risk, or hospital-adapted, lineages. The proposed research concerns the relationship of the recently discovered putative prokaryotic immune system, Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas, with the emergence and continuing persistence of multidrug resistant E. faecalis. Loss of CRISPR-Cas defense may have contributed to the evolution and expansion of high-risk enterococcal lineages by enabling acquisition of new traits, such as antibiotic resistance, that facilitate persistence i clinical environments. The specific aims of this research are to: 1) test the hypothesis that CRISPR-Cas interferes with plasmid acquisition by E. faecalis, 2) characterize requirements for small CRISPR RNA processing, and 3) analyze E. faecalis population structure by CRISPR typing. This research will provide insight into a potential mechanism contributing to antibiotic resistance and virulence gene accretion in E. faecalis, and has implications for the identification of E. faecalis strains safe for industrial use. The bacterium Enterococcus faecalis is among the leading causes of healthcare-associated infections in the United States. Multidrug resistance acquired by horizontal gene transfer is characteristic of high-risk E. faecalis strains. The proposed research will evaluate the role of the putative prokaryotic immune system CRISPR-Cas in control of horizontal gene transfer in E. faecalis.